Study of the spiramycin biosynthesis and its regulation in streptomyces ambofaciens

N°D’ORDRE
UNIVERSITE DE PARIS-SUD XI
UFR SCIENTIFQUE D’ORSAY

THESE
Présentée pour obtenir

LE GRADE DE DOCTEUR EN SCIENCE DE L’UNIVERSITE
DE PARIS-SUD XI

PAR

HOANG CHUONG NGUYEN

SUJET : Study of the spiramycin biosynthesis and its regulation in
Streptomyces ambofaciens

DIRECTEURS DE THESE

Pr. Thuy Duong HO HUYNH
Dr. Jean-Luc PERNODET

Soutenue publiquement le 29 Septembre 2009

JURY

Pr. Armel GUYONVARCH
Pr. Pierre LEBLOND
Pr. Florence MATHIEU
Pr. Philippe JACQUES
Dr. Jean-Luc PERNODET

Pr. Thuy Duong HO HUYNH

Président
Rapporteur
Rapporteur
Examinateur
Examinateur
Examinateur


N°D’ORDRE
UNIVERSITE DE PARIS-SUD XI
UFR SCIENTIFQUE D’ORSAY

THESE
Présentée pour obtenir

LE GRADE DE DOCTEUR EN SCIENCE DE L’UNIVERSITE
DE PARIS-SUD XI

PAR

HOANG CHUONG NGUYEN

SUJET : Study of the spiramycin biosynthesis and its regulation in
Streptomyces ambofaciens

DIRECTEURS DE THESE

Pr. Thuy Duong HO HUYNH

Dr. Jean-Luc PERNODET

Soutenue publiquement le 29 Septembre 2009

JURY
Pr. Armel GUYONVARCH
Pr. Pierre LEBLOND
Pr. Florence MATHIEU
Pr. Philippe JACQUES
Dr. Jean-Luc PERNODET
Pr. Thuy Duong HO HUYNH

Président
Rapporteur
Rapporteur
Examinateur
Examinateur
Examinateur

1



Remerciements
J’ecris ces remerciements en Français parce que, simplement, les gens auxquels je tiens à
exprimer ma gratitude sont français ou bien qu’ils connaissent bien la langue française. En
plus, je trouve que le français est une langue qui convient bien pour exprimer des
sentiments.

La plupart de ce travail a été réalisé dans le Laboratoire de Microbiologie Moléculaire des

Actinomycètes de l’Institut de Génétique et Microbiologie, UMR CNRS 8621 en collaboration
avec le Laboratoire de Génétique de l’Université des Sciences Naturelles à Ho Chi Minh Ville
dans le cadre d’une thèse en co-tutelle.
Tout d’abord, je suis très reconnaissant à mes directeurs de thèse, Madame Thuy Duong Ho
Huynh et Monsieur Jean-Luc Pernodet. Madame Ho Huynh a initié cette collaboration, m’a
donné la possibilité de venir travailler en France et m’a aidé pour toutes les démarches
administratives. Elle a toujours suivi mon travail et m’a conseillé. Je tiens à remercier tout
particulièrement Monsieur Pernodet pour la façon dont il a dirigé ma thèse : sa confiance en
moi, sa grande disponibilité, sa générosité, son enthousiasme et ses connaissances qu’il a
partagées avec moi. Grâce à lui, j’ai pu également découvrir pleinement la vie ‘‘à la
française’’ et passer des moments mémorables un peu partout en France, en Angleterre et
en Allemagne.
Je remercie le Professeur Florence Mathieu et le Professeur Pierre Leblond de me faire
l’honneur d’accepter d’être rapporteurs de cette thèse.
Je remercie le Professeur Philippe Jacques et le Professeur Armel Guyonvarch d’avoir
accepté de participer à ce jury d’examen.
Je tiens à remercier mes deux tuteurs de thèse, le Professeur Armel Guyonvarch et le
Docteur Philippe Mazodier pour avoir suivi ce travail et pour leurs conseils.
Je remercie aussi le conseiller aux thèses, le Docteur Marie-José Daboussi et le directeur de
l’école doctorale GGC, le Professeur Giuseppe Baldacci pour leurs encouragements, le suivi
de mon travail et leur soutien.

2



J’exprime mes profonds remerciements à tous les membres passés ou présents du
Laboratoire de Microbiologie Moléculaire des Actinomycètes, du Laboratoire de Métabolisme
Energétique des Streptomyces et de l’institut de Génétique et Microbiologie : Fatma Karray
pour m’avoir guidé quand j’ai débuté mon travail de thèse, Sylvie Lautru pour sa grande aide

aussi bien pour les expériences, en particulier celles de chimie analytique, que pour la
rédaction, Muriel Decraene pour toutes les formalités françaises compliquées pour lesquelles
je ne sais pas me débrouiller sans elle, et aussi tous les autres pour leurs conseils, leur
sympathie et leur soutien, Karine Tuphile, Annick Friedmann, Alain Raynal, Michel Cassan,
Claude Gerbaud, Emmanuelle Darbon-Rongère, Marie-Hélène Blondelet-Rouault, Josette
Gagnat, Catherine Esnault, Marie-Joëlle Virolle, Sylvain Pendino, Cécile Martel, Jean-Denis
Le Manach, Thierry Locatelli.
Je tiens également à remercier les jeunes des labos, Sarka, Maud, Nicolas, Céline,
Aleksandra, Noriyasu, Aleksei, Hanane, Amélie, Fabien, Emilie, Florence, Hasna, Delin qui
ont suivi au fil de ma thèse mes espoirs aussi bien que mes découragements et m’ont fait
bénéficier de leur aide, leur soutien constant et leur bonne humeur.
Ces quatre dernières années ont été riches en moments de convivialité. Les nombreux
barbecues, les sorties (culturelles et/ou gastronomiques) et les soirées de fêtes resteront des
souvenirs inoubliables.

3



REMERCIEMENTS

2

Chapter I: INTRODUCTION
1. The genus Streptomyces
1.1.Taxonomy
1.2. The genome of Streptomyces
1.2.1. Streptomyces chromosomes
1.2.2. Streptomyces extrachromosomal elements
1.2.3. Streptomyces phages

1.3. Morphological development of Streptomyces
1.4. Ecology

6
6
6
8
8
12
13
13
18

2.The biosynthesis of secondary metabolites by Streptomyces
and related actinobacteria.
2.1. The importance of secondary metabolites for medicine
and agriculture
2.2. Biological activities of secondary metabolites
from Streptomyces and related actinobacteria
2.2.1. Antibacterial and antifungal agents
2.2.2. Antitumor agents
2.2.3. Enzymes inhibitors
2.2.4. Immunosuppressants
2.2.5. Insecticides and antiparasitic drugs
2.2.6. Herbicides
2.3. The genetics of secondary metabolism in Streptomyces
2.3.1. Initial genetic studies of secondary metabolism in S. coelicolor
2.3.2. The isolation of secondary metabolites biosynthetic gene clusters in Streptomyces
2.4. The biosynthesis of polyketides
2.4.1. The assembly-line enzymology of polyketide synthases

2.4.2. The different types of PKSs
2.4.2.1. Type I PKS
2.4.2.2. Type II PKS
2.4.2.3. Type III PKS
2.5. The biosynthesis of nonribosomal peptides
2.6. The regulation of secondary metabolism in Streptomyces
2.6.1. Multiple factors influence the onset of secondary metabolism
2.6.2. Gowth rate and nutriment limitation
2.6.3. Diffusible signalling molecules
2.6.4. Regulatory proteins controlling secondary metabolism in Streptomyces

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25
25
26
27
28
29
29
29
31
31
33
33
36
37
38
40
40

41
43
45

3. Macrolide antibiotics and their biosynthesis in Actinobacteria
3.1. Definition and classification of macrolide antibiotics
3.2. The mode of action of macrolide antibiotics
3.3. Resistance to macrolide antibiotics
3.3.1. Target modification
3.3.2. Efflux of macrolides
3.3.3. Inactivation of macrolides
3.4. Biosynthesis of macrolides
3.4.1. Biosynthesis of the macrolactone ring
3.4.2. Biosynthesis of the sugars
3.4.3. Tailoring reactions
3.4.4. Enzymes involved in glycosylation during macrolide biosynthesis
3.5. Combinatorial biosynthesis of new macrolide antibiotics
3.5.1. Combinatorial biosynthesis of the macrolactone ring
3.5.2. Modification of the glycosylation pattern through combinatorial biosynthesis
3.6. The regulation of macrolide biosynthesis
3.6.1. Regulation of erythromycin biosynthesis
3.6.2. Regulation of methymycin/pikromycin biosynthesis
3.6.3. Regulation of tylosin biosynthesis
3.7. Spiramycin biosynthesis in S. ambofaciens

47
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50
52
53

54
54
55
55
57
58
60
62
63
66
68
68
69
70
72

Chapter II: GLYCOSYLATION STEPS DURING SPIRAMYCIN BIOSYNTHESIS
IN STREPTOMYCES AMBOFACIENS: INVOLVEMENT OF THREE
GLYCOSYLTRANSFERASES AND TWO AUXILIARY PROTEINS

75

Chapter III: A POST-PKS PLATENOLIDE KETOREDUCTASE IS INVOLVED
IN SPIRAMYCIN BIOSYNTHESIS IN STREPTOMYCES AMBOFACIENS

103

Chapter IV: REGULATION OF SPIRAMYCIN BIOSYNTHESIS
IN STREPTOMYCES AMBOFACIENS

122

4

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22



Chapter V: TRANSCRIPTIONAL ORGANIZATION OF THE SPIRAMYCIN CLUSTER
AND ACTIVITIES OF THE TRANSCRIPTIONAL ACTIVATORS SRMR AND SRMS

151

GENERAL CONCLUSIONS

176

RESUME DE LA THESE EN FRANÇAIS

182

REFERENCES

200

5

Chapter I
Introduction



1. The genus Streptomyces.
1.1. Taxonomy
Streptomyces

are

Gram-positive

bacteria.

They

belong

to

the

family

of

Streptomycetaceae that comprises also the genera Kitasatospora, Parastreptomyces,
Streptacidiphilus, and Trichotomospora. Streptomycetaceae is the only family of the suborder
Streptomycineae that belongs to the order Actinomycetales (Figure 1) (Stackebrandt et al.,

1997). This order belongs to the class of Actinobacteria, whose members are defined as
Gram-positive bacteria with a high G+C content in their DNA.

Figure 1: Phylogenetic relatedness of the families of the class Actinobacteria. Interclass
relatedness of Actinobacteria, showing the orders and the 10 suborders of the order
Actinomycetales, is based upon rDNA/rRNA sequence comparison. The scale bar represents
5 nucleotide substitutions per 100 nucleotides. From (Stackebrandt et al., 1997).
Actinobacteria, which constitute one of the largest bacterial phyla, include
microorganisms exhibiting a wide spectrum of morphologies (coccoid: Micrococcus; rodcoccoid: Arthrobacter, fragmenting hyphal forms: Nocardia; highly differentiated branched
mycelium: Streptomyces) and possessing highly variable physiological and metabolic
properties. Various lifestyles are also encountered among this class, which includes

6


pathogens (e.g. Mycobacterium spp., Corynebacterium spp., Tropheryma whipplei), soil
inhabitants (e.g. Streptomyces), plant commensals (Leifsonia spp.), nitrogen-fixing symbionts
of plants (Frankia) and gastrointestinal tract bacteria (Bifidobacterium spp.).
Several actinobacterial genomes have been sequenced and the diversity of
Actinobacteria is also visible in the characteristics of these genomes (Table 1). The sizes of
these genomes vary from less than 1 Mb (Tropheryma whipplei) to more than 10 Mb
(Streptomyces scabies). Most actinobacterial genomes are circular, but the genomes of
Streptomyces are linear. The G+C content is generally high, from 53% for Corynebacterium
to over 70% for Streptomyces, but it is of only 46% for the small genome of the obligate
pathogen Tropheryma whipplei.
Microrganism

Genome Number of % G+C Circular Reference
size (Mb) genes
(C)

or Acc. N°
linear (L)

Bifidobacterium longum

2.26

1 797

60

C

NC_004307

Corynebacterium diphtheriae

2.48

2 341

54

C

NC_002935

Corynebacterium glutamicum

3.14

3 128

54

C

NC_009342

Frankia alni

7.49

6 774

72

C

NC_008278

Leifsonia xyli subsp. xyli

2.58

2 376

68

C

AE016822

Micrococcus luteus

2.50

2 236

72

C

NC_012803

Mycobacterium leprae

3.26

1 655

58

C

AL450380

Mycobacterium tuberculosis

4.41

4 039

66

C

NC_000962

Nocardia farcinica

6.02

5 747

71

C

NC_006361

Propionibacterium acnes

2.56

2 351

60

C

AE017283

Rhodococcus erythropolis
PR4

6.52

6 104

62

C

NC_012490

Rhodococcus jostii. RAH1

7.80

7 274

67

L

NC_008268

Saccharopolyspora
erythreaea

8.21

7 259

7

C

NC_009142

Streptomyces avermitilis

9.02

7 669

71

L

BA000030

Streptomyces coelicolor

8.66

7 855

72

L

NC_003888

Streptomyces griseus

8.54

7 222

72

L

NC_010572

7

or


Streptomyces scabies

10.14

Not
yet 72
annotated

L

www.sanger.ac.
uk/Projects/S_s
cabies/

Thermobifida fusca

3.62

3 177

67

C

NC_007333

Tropheryma whipplei

0.92

838

46

C

NC_004551

Table 1: Some characteristics of the genome from selected representatives of the
Actinobacteria.

Among Actinobacteria, the genus Streptomyces groups mycelial spore-forming
bacteria and identified by both chemotaxonomic and phenotypic characters including 16S
rRNA homologies, cell wall analysis, fatty acid and lipid patterns (Wellington et al., 1992;
Williams et al., 1989). Streptomycetes are believed to have originated about 440 millions
years ago (Embley & Stackebrandt, 1994).

1.2. The genome of Streptomyces
The genome of Streptomyces is remarkable for several reasons: its size, its high G+C
content, the linearity of the chromosome and the organization of the genes on the
chromosome. Moreover, some of type of accessory genetic elements has so far been found
only in Actinobacteria.
1.2.1. Streptomyces chromosomes
The Streptomyces chromosomes have sizes in the range 8-10 Mb (see table 1). This
is about twice the size of the chromosome of bacteria such as Escherichia coli or Bacillus
subtilis. The number of genes carried by this chromosome is also quite large and S.
coelicolor (7 855 genes) was the first example of a bacteria possessing more genes than a
unicellular eukaryote such as Saccharomyces cerevisiae (6 023 genes).
The average G+C content of Streptomyces chromosomal DNA is in the range 70-72
% (Table 1). In coding sequences, the codon usage is strongly biased towards codons with C
or G in the third position; this bias is used to predict probable coding sequences among all
possible open reading frames (Wright & Bibb, 1992).
The linearity of the Streptomyces chromosome was first established for S. lividans
(Lin et al., 1993) and then demonstrated in other species including S. ambofaciens (Leblond
& Decaris, 1994) and S. coelicolor (Redenbach et al., 1996). It is now considered that all
Streptomyces have a linear chromosome, but this is not true for all related mycelial
actinobacteria with a large genome, as for instance Saccharopolyspora erythraea, which has

8


a circular chromsome (Oliynyk et al., 2007). Linearity has not been apparent from the circular
genetic linkage map of S. coelicolor (Hopwood, 1967). The Streptomyces linear chromosome
possess terminal inverted repeats (TIR), the size of which varies from 174 bp for S.
avermitilis (Ikeda et al., 2003)

to several hundred of kilobases, e.g. 198 kb for S.

ambofaciens (Choulet et al., 2006) and about 550 kb for S. rimosus (Pandza et al., 1997).
The chromosome is replicated bidirectionally from the oriC located in the centre of
chromosome towards the telomeres at the two extremities (Jakimowicz et al., 2000). A
protein (terminal protein, TP) is covalently bound to both free 5’ ends. This terminal protein,
together with a telomere associated protein (TAP), is required for the synthesis of the last
Okazaki fragment of the lagging strand when bidirectional DNA replication reaches the free
ends.
A

remarkable

feature

of

Streptomyces

chromosomes

is

their

highly

compartmentalized genetic organization. This was already apparent with the sequence of the
S. coelicolor chromosome, where all house-keeping genes were located in the central part
(core region) of the chromosome (Figure 2). In S. coelicolor no essential gene, with the
single exception of argG, is located within 1.3 Mb of each extremity.

9


Figure 2: Representation of the S. coelicolor chromosome. The outer scale is numbered
anticlockwise in megabases and indicates the core (dark blue) and arm (light blue) regions of
the chromosome. Circles 1 and 2 (from the outside in), all genes (reverse and forward
strand, respectively) colour-coded by function (black, energy metabolism; red, information
transfer and secondary metabolism; dark green, surface associated; cyan, degradation of
large molecules; magenta, degradation of small molecules; yellow, central or intermediary
metabolism; pale blue, regulators; orange, conserved hypothetical; brown, pseudogenes;
pale green, unknown; grey, miscellaneous); circle 3, selected 'essential' genes (for cell
division, DNA replication, transcription, translation and amino-acid biosynthesis, colour
coding as for circles 1 and 2); circle 4, selected 'contingency' genes (red, secondary
metabolism; pale blue, exoenzymes; dark blue, conservon; green, gas vesicle proteins);
circle 5, mobile elements (brown, transposases; orange, putative laterally acquired genes);
circle 6, G + C content; circle 7, GC bias ((G - C/G + C), khaki indicates values >1, purple
<1). The origin of replication (Ori) and terminal protein (blue circles) are also indicated. From
(Bentley et al., 2002).

The comparative analysis of Streptomyces chromosome sequences revealed that the

core regions are highly syntenic and contain most of the genes conserved with other
Actinobacteria while the subtelomeric regions are species-specific (Bentley et al., 2002;
Choulet et al., 2006; Ikeda et al., 2003). This is illustrated by the comparison of the S.
coelicolor and S. avermitilis chromosomes presented in figure 3.

10


Figure 3: Synteny between S. avermitilis and S. coelicolor A3(2) linear chromosomes. Each
point in this figure is a reciprocal best hit. These hits were obtained by pairwise BLASTP
searches of predicted S. avermitilis proteins against those of S. coelicolor A3(2). Each
protein pair is graphed according to the location of the corresponding gene on respective
DNA molecules. The bars above and at right of plot indicate the region conserved to circular
Actinobacterium chromosomes (green), subtelomeric (blue), and backbone (red) regions in
S. avermitilis and S. coelicolor A3(2), respectively. Arrows indicate the position of oriC. A, B,
and C indicate inverted regions between S. avermitilis and S. coelicolor A3(2). From (Ikeda
et al., 2003).

Comparison of the chromosome of S. avermitilis with those of the two closely related
species S. coelicolor and S. ambofaciens revealed that the size of the central region
conserved between species decreases as the phylogenetic distance between them
increases, whereas the specific terminal fraction reciprocally increases in size (Choulet et al.,
2006). Between highly syntenic central regions and species-specific subtelomeric regions,
there is a notable degeneration of synteny due to frequent insertions/deletions.
These terminal regions also suffer large deletions and amplifications which can occur
spontaneously with high frequency (Altenbuchner & Cullum, 1985; Chen et al., 2002;
Schrempf et al., 1989). Regions of more than several hundreds of kilobases can be deleted

11

in the terminal parts without any deleterious effects on the viability of Streptomyces strains in
laboratory conditions (Volff & Altenbuchner, 1998). Interestingly, deletions affecting the
terminal regions can lead to chromosome circularization (Catakli et al., 2003; Lin et al., 1993;
Redenbach et al., 1993).
1.2.2. Streptomyces extrachromosomal elements
Many Streptomyces strains contain plasmids, most of which are phenotypically
cryptic. These plasmids can be circular or linear. Linear plasmids have sizes in the range of
10 to 600 kb. As the chromosome, they have a centrally located replication origin, terminal
inverted repeats and depend for their replication on a covalently bound terminal protein and
on a telomere associated protein (Chater & Kinashi, 2007). Among circular plasmids, the
larger ones (e.g. SCP2, 31 kb) have a low copy number and replicate bidirectionally by a θ
mode of replication. The smaller plasmids (e.g. pIJ101, 8.9 kb) tend to have higher copy
numbers and replicate by a rolling circle mechanism via a circular, single-stranded replication
intermediate. Their replication is initiated by a plasmid-encoded replication protein (Kieser et
al., 2000a). Plasmid cloning vectors have been developed mostly from small circular
plasmids, but some vectors are derived from SCP2.
A novel type of mobile genetic element was first discovered in Streptomyces and later
in some other Actinobacteria: the actinomycete integrative and conjugative elements (AICEs)
(te Poele et al., 2008). These genetic elements belong to a broad class of integrative and
conjugative elements (ICEs) (Burrus et al., 2002), which have both plasmid-and
bacteriophage-like features. ICEs are normally integrated in the host chromosome, but they
have the ability to excise, conjugate to a new host and integrate in the new host chromosome
by site-specific recombination. AICEs represent a special class of ICEs, because unlike other
ICEs, they have the ability to replicate autonomously like a plasmid. For some of the AICEs,
the integrated and replicative forms can even coexist. Most of the AICEs integrate in a
specific tRNA gene in the host chromosome and this gene is not inactivated after integration
because the AICE and the chromosome share a segment of identity (Mazodier et al., 1990).
The most studied representatives of the AICEs are SLP1 from S. coelicolor (Bibb et al.,
1981), pSAM2 from S. ambofaciens (Pernodet et al., 1984) and pMEA300 from

Amycolatopsis methanolica (Vrijbloed et al., 1994). As they are able to replicate and to
integrate site-specifically, AICEs have been used to develop cloning vectors (Cohen et al.,
1985; Smokvina et al., 1990; Vrijbloed et al., 1995).
Most of the plasmids and all of the AICEs are conjugative mobile genetic elements.
But the conjugation mechanism in Streptomyces is very different from the one in other
bacteria, the paradigm of which is the transfer mechanism of the F sexual factor from

12


Escherichia coli. For the transfer of F, the products of about 30 transfer genes are required
(Frost et al., 1994). These gene products first establish a stable mating pair via cell-to-cell
junctions. DNA transfer is initiated by the relaxase that nicks DNA at the origin of transfer
(oriT) to generate a single-stranded molecule. The resulting structure, the relaxosome, is
then coupled to the transferosome (related to type IV secretion systems) by the intermediate
of a coupling protein. In Streptomyces, the conjugation machinery is actually much simpler
with a single membrane-associated ATPase (Tra) similar to DNA translocases, such as
SpoIIIE of Bacillus subtilis (Wu & Errington, 1997), sufficient for plasmid transfer (Reuther et
al., 2006). A plasmid-borne cis-acting sequence of about 50 bp, called clt, is recognized by
Tra (Reuther et al., 2006). Another original feature is the transfer of double-stranded DNA
(Possoz et al., 2001). It was confirmed recently that no DNA cleavage occurs at the clt locus
(Ducote & Pettis, 2006). The protein Tra is located at the tip of the growing hyphae (Reuther
et al., 2006). Conjugative mobile genetic elements are also able to mobilize chromosomal
gene markers, but in contrast to the recent knowledge on plasmid transfer in Streptomyces,
chromosomal DNA mobilization is very poorly documented, except that it was proven that
conjugative elements stimulate chromosomal DNA transfer (Pettis & Cohen, 1994).
1.2.3. Streptomyces phages
Most of Streptomyces phages have been isolated from soil because nearly all soil
samples readily yield phages that give plaques on S. lividans 66, a generally permissive host
for Streptomyces phages rare (Kieser et al., 2000a). Besides, Streptomyces themselves

provide a second source of phages because some phages have been discovered after
release by natural lysogenic strains. Phages isolated from soil have generally a wide host–
range. In contrast, phages isolated from lysogenic strains have generally a narrow hostrange. Nearly all the Streptomyces phages examined have polyhedral heads and long tails
As with other bacteriophages of similar morphology, they contain double-stranded DNA
(Lomovskaya et al., 1980). Streptomyces phages are both lytic and temperate and it is
infrequent that Streptomyces phages infect other genera of actinomycetes. Most phages are
active on germ tubes and young mycelium but some can be active on old mycelium.

1.3. Morphological development of Streptomyces
Streptomyces are mycelial spore-forming bacteria, remarkable for their morphological
development and considered to be among the most complex of bacteria (Chater & Chandra,
2006; Flardh & Buttner, 2009). A spore germinates to give a vegetative mycelium. Then
aerial hyphae, which are at least partially parasitic on the vegetative mycelium, emerge.

13


These aerial hyphae finally differentiate to form chains of spores. These steps are presented
in Figure 4 and the resulting developmental cycle is represented in Figure 5.

Figure 4: Mycelial forms during the development of Streptomyces. From (Flardh & Buttner,
2009).

Figure 5: Life cycle of Streptomyces.
(From />
This morphological development has been studied, especially in Streptomyces
coelicolor and Streptomyces griseus, and many genes playing a role in development have

14

been identified. Much of these genes were identified by the isolation and characterization of
S. coelicolor mutants that failed complete normal development and were affected either in
aerial growth (“bald” or bld mutants) or in the formation and maturation of spores (“white” or
whi mutants). Most of the genes thus identified are regulatory genes and their interactions
have been partly characterized. The different steps of the developmental cycle and the major
genes involved are detailed below.
When a spore encounters favourable conditions, it germinates and one or two germ
tubes emerge. These germ tubes grow as thread-like hyphae by tip extension and branching
to form a vegetative or substrate mycelium. This mycelium often goes deep into the
surrounding substrate. New cell wall material is synthesized only at the hyphal tip. This
process involves the coiled-coil protein DivIVA. DivIVA acts as a landmark protein that
recruits directly or indirectly the cell wall biosynthetic machinery. The hyphal tip is probably
also a hot spot where peptidoglycan, teichoic acid, cell-surface proteins, and membrane
lipids are secreted and assembled. Apart from DivIVA, there are other proteins residing at
the hyphal tip regions and participating in the apical growth such as the cellulose synthaselike protein CslA which interact with DivIVA to add a so far uncharacterised β-linked glucan in
the cell envelope, or, in a later stage, the surfactant protein SapB (spore-associated protein
B) which has a function in the aerial mycelium development.
In response to nutrient depletion and other signals, the morphological differentiation is
initiated and the formation of aerial mycelium starts. This is often accompanied by
modifications at the metabolic level and the commitment to secondary metabolism. The
aerial mycelium breaks the surface tension, escaping the aqueous environment of substrate
mycelium and grows into the air. To do that, Streptomyces has to coat its aerial hyphae in a
hydrophobic sheath that is absent from the substrate mycelium. The SapB protein is
produced to allow efficient formation of aerial mycelium. This protein is only produced on rich
media. On minimal media, other proteins replace SapB to be the major components of the
hydrophobic sheath. They are called the chaplins and the rodlins. Streptomyces strains that
cannot make both SapB and chaplins are bald, i.e. they cannot form aerial mycelium, under
all growth conditions. When studied in vitro, both SapB and chaplins have powerful
surfactant activities at air-water interface. They lower the surface tension from 72 mJ per m2

to 27 mJ per m2. The rodlin proteins have less impact on the formation of aerial mycelium.
Mutants that lack rodlin proteins still form a hydrophobic sheath and aerial mycelium. Instead
of having normal surface ultrastructure, the rodlin mutants have a disordered network of fine
filaments. The S. coelicolor bld mutants are blocked in aerial mycelium formation, but when
different mutants are grown near to each other on certain media, most of them exhibit
directional interactions that lead to aerial mycelium formation. These interactions have been

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interpreted as resulting from the existence of an extracellular signalling cascade. The
signalling molecules are unknown. This cascade is represented in Figure 6.

Figure 6: Extracellular signalling cascade dependent on bld genes in S. coelicolor. From
(Chater & Chandra, 2006).

In other Streptomyces species, different extracellular interactions controlling aerial
mycelium formation have been described. In particular, in S. griseus, A-factor, a compound
of the gamma-butyrolactone family, is a extracellular signal required for aerial mycelium
formation but also for the switch to the synthesis of the antibiotic streptomycin.
The last step of the cycle is the differentiation of aerial hyphae into spores (Figure 7).
It starts with the formation of a long, non-septated, apical compartment, called sporogenic
cell, each of which contains 50 or more copies of chromosome. Then, the sporogenic cell
stops its extension and begins synchronous, multiple cell division, under the effect of the
whiA and whiB gene products. The next step is the formation of septum, directed by the
bacterial tubulin homologue FtsZ. In virtually all bacteria, FtsZ assembles into a cytokinetic
ring, the Z ring that defines the site of division and recruits other cell-division proteins. During
the stage of septation, DNA is segregated into unigenomic spores, but complete partitioning
into individual nucleoids does not occur until the final stage of septation. The DNA
segregation involves at least two systems: ParAB and FtsK. Sporulation septa constrict over

unsegregated nucleoids. Finally, a thick, lysozyme-resistant, spore wall is formed. This wall
is laid down after sporulation septation is complete and is associated with the rounding up of
the cylindrical prespore into an ovoid shape. This step involves MreB. Then, one cycle is
finished and the spore can be disseminated and give rise to a new colony.

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Figure 7: Aerial hyphae differentiation into spore chains in S. coelicolor. From (Chater &
Chandra, 2006).

Aerial growth is partly parasitic on the primary colony, which is digested and reused
for aerial growth. This late phase of development is coordinated with the production of
antibiotics, which may protect the colony against invading bacteria during aerial growth.
Among Actinobacteria, the Streptomycetes are the only ones for which development
was studied in detail and many of the genes involved in aerial mycelium development and
spore formation have been identified. A comparative genomic analysis suggests that the
evolution of Streptomyces development has probably involved the stepwise acquisition of
laterally transferred DNA. Each of these acquisitions gave rise to regulatory changes or to
changes in cellular structure and morphology (Figure 8) (Chater & Chandra, 2006; Ventura
et al., 2007).

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